Purification of somatotropin from transformed microorganisms

ABSTRACT

This invention discloses a commercially advantageous process for extraction and purification of protein from microorganisms. The initial steps of the process are useful for purifying many insoluble proteins while later steps are designed to renature denatured somatotropins produced by transformed microorganisms. The process is especially useful for purifying recombinantly-produced bovine somatotropin.

This application is a continuation-in-part of U.S. Ser. No. 06/749,016,filed Jun. 26, 1985, now abandoned.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

This invention discloses a commercially advantageous process forextraction and purification of protein from microorganisms. The initialsteps of the process are useful for purifying many insoluble proteinswhile later steps are designed to renature denatured somatotropinsproduced by transformed microorganisms. The process is especially usefulfor purifying recombinantly-produced bovine somatotropin (rbSt).

Somatotropins are growth hormones which were originally discovered inpituitary gland extracts of various animals. In general, somatotropinsare conserved molecules and similarities in amino acid sequence andstructure are found between animals of disparate evolutionary rankingevidencing a common ancestral relationship.

Somatotropins are globular proteins comprised of a single chain ofapproximately 200 amino acids, having 2-3 intrachain disulfide bonds.Accordingly, bovine somatotropin (bSt) is comprised of a single chain of190-191 amino acids, a globular structure with two intrachaindisulfides, and a molecular weight of approximately 22,000 daltons.

BSt is able to remain insoluble in an aqueous environment when in thereduced state. This characteristic is exploited in the present inventionas it relates to extraction and purification procedures. The disclosedprocess is applicable for other somatotropins having this samecharacteristic.

Growth hormones especially bSt have great commercial potential. Growthhormones in general have been shown to increase the growth rate ofcattle, hogs and salmon, along with a significant increase in the meatto fat ratios. Moreover, bSt has value in its proven ability to increasemilk production in dairy cattle. It is presently anticipated that 5-50mg of growth hormone will be necessary per day per animal to achieveeffective results.

Although a number of processes are presently available for theextraction of somatotropins from E. coli or other microorganisms on alaboratory scale, a process for the commercial production or large scaleproduction of bioactive somatotropins, especially bST, was not availableup until now. The disclosed process optimizes recovery of somatotropinsfrom a microorganism when that microorganism has produced thesomatotropin in predominantly precipitated form rather than as a solubleprotein. These precipitated somatotropins are often deposited inrefractile bodies within the microorganism. The refractile bodies arevisible under a light microscope.

Somatotropins are not the only heterologous proteins to form retractilebodies inside microorganisms. Refractile bodies are often produced as aresult of overproduction of a protein. They are thought to be aninternal storage mechanism. Refractile bodies are commonly seen inmicroorganisms expressing heterologous proteins due to pathogenicconditions, mutations or recombinant genetic transformation.

Precipitated somatotropins are thought to be in a reduced state whenfound in refractile bodies of transformed microorganisms such as E.coli. Although expression of eukaryotic protein in E. coli or otherprokaryote systems can yield a amino acid chain identical to the naturalproduct often times the secondary and tertiary structure of the nativeprotein cannot be forced or duplicated by the prokaryote. Unless theenergy state of the native form forces the product to the nativeconformation, chemical and thermal intervention will be necessary toyield a biologically active and non-antigenic product.

BSt has 4 cysteine residues for which 3 different arrangements ofdisulfide bonds are possible in the oxidized monomeric state, only oneof which is native. Upon workup without the precautions disclosed hereinthe cysteine residues will oxidize using available oxygen to form bothpolymeric and monomeric bSt with random disulfides. The random states sogenerated result in substantial yield reductions of bioactive growthhormones.

The classical studies of protein folding involving reduction andreoxidation of proteins were performed on enzymes such as ribonuclease(RNAse) and lysozyme. Givol, D., DeLorenzo, F., Goldberger, R. F., andAnfinsen, C. B., Biochemistry 53:676-684 (1965); and Saxena, V. P., andWetlaufer, D. B., Biochemistry 9(25):5015-5022 (1970). The proteins aretypically reduced with B-mercaptoethanol (BME) or dithiothreitol (DTT)in the presence of a denaturant such as 6-8 M urea or guanidine-HCl. Thereductant and denaturant is then removed by gel filtration, and theprotein is allowed to air-oxidize at low concentration.

The mechanism presently accepted for the reoxidation of reduced proteinsinvolves rapid reoxidation of the protein to form random disulfidebonds, followed by disulfide interchange (“shuffling”). Although theprotein may form incorrect disulfide bonds at first, these disulfidesmay be reduced again by reaction with free thiol groups on otherproteins or thiol reagents. This gives each protein molecule the chanceto assume a variety of disulfide bonding modes until the majority of themolecules are in the lowest energy configuration. The lowest energystate is usually the native conformation. Acharya, A. S., and Taniuchi,H., Molec. and Cell. Biochem. 44:129-148 (1982); and Anfinsen, C. B.,Science 181:223-230 (1973).

Unlike the proteins described above, fully reduced bSt is insoluble inmost aqueous solvents. Thus, many of the classical techniques foroxidation of reduced proteins are not applicable to bSt and theavailable methods taught in the literature concerning rbSt purificationfrom E. coli were not practical for commercial applications.

The available literature discloses general principles of purification ofheterologous proteins from microorganisms. Basically the steps involvecell kill, lysis, selective solubilizing of host debris, mechanicalcollection of the precipitated heterologous protein and solubilizing theheterologous protein followed by further filtration steps.

Prior to this invention, it was not possible to kill host cells withouta concomitant precipitation of large amounts of undesired protein andother host cell debris. The precipitation of undesired. protein alongwith desired insoluble proteins adds to the complexity of downstreampurification procedures. This invention discloses the use of nonpolarorganic solvents to kill cells. By eliminating standard killing stepsusing heat, phenol or combinations of phenol and toluene and by usingthe nonpolar organic solvents disclosed herein, it is possible tominimize unwanted host protein precipitation.

This invention also provides for an effective one step renaturation ofsomatotropins in the presence of detergents. Prior art recommendationssuggest that renaturing of any desired protein to its native state hadto be done at low concentrations of less than 1 mg/ml to avoidpolymerization of bSt through cysteine residues. This invention resolvesthat problem by avoiding solubilization of somatotropins in nondetergentchaotropic agents such as urea or guanidine. By using mild detergents tosolubilize the somatotropins, this invention demonstrates that it ispossible to renature rbSt and like somatotropins at concentrationsgreater than 15 mg/ml.

The disclosed process has other advantages over the prior art processes.This process eliminates the need for thiol-reducing agents such asmercaptoethanol or glutathione. There is no need to use highconcentrations of denaturants such as urea or guanidine to solubilizethe desired proteins. Urea and guanidine have to be used at molarconcentrations of up to 9 moles per liter and such concentrations willinevitably cause problems in large scale purifications resulting inincreased production costs. This procedure also provides foralternatives to sodium dodecyl sulfate (SDS) as a solubilizing detergentwhich are advantageous because SDS is known to be difficult to removefrom bSt. Dellacha, J. M., Annals N.Y. Acad. Sci., 148:313-327 (1968).One further advantage of this procedure is that bSt is split into twodiscrete populations upon removal of detergent. Upon removal ofdetergent, rbst is found as either a soluble monomer or as an insolubleaggregate which is readily removable. The proportion of dimers ortrimers is not significant and their removal by gel filtration or by ionexchange chromatography is optional.

Lastly, the disclosed process is more economical when compared toalternative purification procedures.

Using the disclosed procedure, yields of up to 60% of the rbSt producedby the organism can be obtained. The rbSt is in the native and bioactiveform as determined by in vivo tests in hypophysectomized rats. Parlow,A. F., et al., Endocrin. 77:1126 (1965).

Information Disclosure

Expression of somatotropins from a variety of animals by transformedmicroorganisms is known. Goeddel, D. V. et al, “Direct Expression inEscherichia coli of a DNA sequence coding for human growth hormone”,Nature 281, 544-548 (1979) and Seeburg, P. H. et al, “EfficientBacterial Expression of Bovine and Porcine Growth Hormones”, DNA 2:37-45(1983).

Naturally occuring bSt is a mixture of heterogeneous proteins, the aminoacid sequences of which are known. Paladini, A. C., et al., MolecularBiology of Growth Hormone, CRC Reviews in Biochem., 15(1):25-56 (1983).The naturally occurring mixtures have been purified from pituitaryglands of cattle. The commercial potential is well recognized anddocumented by biological studies on both dairy and feed cattle. Eppard,P. J. and Bauman, D. E., The Effect of Long-Term Administration ofGrowth Hormone on Performance of Lactating Dairy Cows; and Bauman,. D.E., Effect of Growth Hormone on Growth Rates and Mammary Development ofRuminants, Proc. 1984 Cornell Nutrition Conference for FeedManufacturers, pp. 5-17, published by Cornell University, Ithaca, N.Y.

The production of bSt in transformed microorganisms can be achieved by avariety of recombinant genetic plasmids. University of California, DNATransfer Vectors Coding for Bovine Growth Hormone, . . . , and Usefulfor Transforming Microorganisms for Production of Fusion Proteins,European Patent Application 47 600; Yeda Research and DevelopmentCompany, Production of Bovine Growth Hormone by Microorganisms; UnitedKingdom Patent Application; GB 2073245A; and Schoner, B. E. et al., Roleof mRNA Translational Efficiency in Bovine Growth Hormone Expression inEscherichia coli, PNAS USA, 81:5403-5407 (1984).

Analogs of bSt are also known. Biogen NV, DNA Sequences, Recombinant DNAMolecules and Processes for Producing Bovine Growth Hormone-LikePolypeptides in High Yield, European Patent Application 103 395; andSchoner, B. E., et al., Supra. Unlike the present invention theseanalogs of bSt relate to the insertion of bases at the 5′ and 3′ ends ofbSt creating a protein different from the naturally-occurring amino acidsequences.

The production of bSt in transformed microorganisms other than E. colihas been reported. Gray, G., et al., Synthesis of Bovine Growth Hormoneby Streptomyces lividans, Gene, 32:21-30 (1984); and in Yeast, U.S. Pat.No. 4,443,539.

The methods of culturing and fermenting the transformed microorganismsis also known and fully described in the above-cited literature.

Purification of biologically active bSt from transformed cells has alsobeen described previously. Genentech, Inc., “Purification and ActivityAssurance of Precipitated Heterologous Proteins”, U.S. Pat. Nos.4,511,502; 4,511,503; 4,512,922 and 4,518,526; Biotechnology GeneralCorp., “Expression Vectors for Enhanced Production of Polypeptides . . .and Related Methods”, European Patent Application 131 843; and, Schoner,R. G., et al., “Isolation and Purification of Protein Granules from E.coli Cells Overproducing bSt”, Bio/Tech., 3:151-154 (1985).

The present invention discloses the use of detergents for placingsomatotropins into a conformation that readily permits proper disulphidebond formation to take place in the presence of the detergent. Uponremoval of the detergent the biologically active state is obtained. Thisis a nonobvious improvement over the Genentech patents which describedetergents as strong denaturants (0.01-2.0%). There the detergents areused to unfold proteins. The detergents are then removed beforeestablishing disulphide bonds and folding the protein into thebiologically active state.

In addition, none of the prior art known to the applicants describes aprocedure having the advantages, efficiency and economic savings of theprocess herein disclosed.

SUMMARY OF THE INVENTION

This invention relates to general processes for extracting insolubleproteins from transformed microorganisms.

The invention specifically relates to an improved procedure to killmicroorganisms prior to the extraction of protein. The cells are exposedto lethal amounts of toluene or xylene or combinations thereof. The useof nonpolar organic solvents is advantageous because such solvents areincapable of substantial protein disruption and denaturation. Unlikephenol, less polar organic solvents such as xylene or toluene willeffectively kill microorganisms without a gross precipitation of hostcontaminants. Where the host cell expresses a substantial proportion ofa desired natural or heterologous protein in a precipitated form, theavoidance of phenol is advantageous in that costs are reduced and higheryields obtained. Once killed the cells are lysed and standard proceduresfor protein extraction and purification are taken to obtain the desiredprotein.

The invention also discloses an advantageous oxidation step downstreamfrom the killing step disclosed above. This step is particularly usefulfor recombinantly produced somatotropins which are insoluble within thecytoplasm of a transformed microorganism. Specifically, this stepdemonstrates that once the majority of host cell protein andcontaminants are removed by the selective solubilization, using weaklydenaturing detergents, the remaining insoluble somatotropins can beoxidized to the biologically active state using dissolved oxygen in astronger solution of detergent. This is a single step conversion to thenative disulphide bond orientation. The reshuffling and refolding stepsdescribed in the prior art are eliminated. The total removal ofdetergent is unnecessary and biological activity of the solution can bedetected after dilution and injection into laboratory animals.Detergents useful for oxidations include sodium dodecyl sulfate (SDS)and those having a structure represented by formula I wherein n is 8-20,R₁ is methyl or ethyl and R₂ is hydrogen, methyl, ethyl, n-propyl orisopropyl. The preferred somatotropin for this step is rbSt.

An advantage of this improvement are the elimination of high molar saltsolutions such as urea or guanidine. Such salts are difficult to handleunder large scale commercial settings and are expensive. In addition,the use of detergents that are easily removed from the growth hormoneallows for increased purity and permits renaturation and oxidation tooccur at concentrations above 5 mg/ml where previous procedures teachthat such concentrations will cause yield-reducing polymerization. U.S.Pat. No. 4,518,526. Finally, oxidation at higher concentrations willresult in lower production cost due to lower process volumes.

In the above processes, the organism of choice is E. coli. The preferredkilling solvent is toluene. The preferred detergent are those having astructure represented by formula 1 wherein the substituents andvariables are as defined above. The most preferred detergent in which tooxidize rbSt to the native state is N-Lauroyl methylglycine.

In the above discussion and throughout this document the terms belowhave the following meanings.

“Biologically-active somatotropins from transformed microorganisms”refers to somatotropin that has been sufficiently restored to its nativeconfiguration that it has detectable activity In mammalian growth ratetests.

“Bovine somatotropin analogs” refer to proteins that are substantiallysimilar to the longest forms of naturally occurring bSt and that havebiological activity useful for increasing milk production and growthrates. Such analogs include both recombinantly produced rbSt having thesame amino acid sequence as the heterogeneous species found in purifiedbSt from pituitary glands and those rbSt species artificially created bymodifying bSt encoding DNA such as described in European patentapplication 103,395.

“Chaotropic agents” refer to protein denaturants which in aqueoussolutions at suitable concentrations are capable of changing the spatialconfiguration or conformation of proteins through alterations at thesurface thereof, either through altering the state of hydration, thesolvent environment or solvent surface interaction. Examples of suchagents include urea, guanidine, hydrochloride, sodium thiocyanate anddetergents such as SDS and Triton X-100.

“Microorganisms” refer to both single cellular prokaryote and eukaryoteorganisms such as bacteria, yeast, actinomycetes and single cells fromhigher order plants or animals when being grown in cell culture asindividual organisms.

“Oxidation” refers to a process in which the reduced thiol groups oncysteine residues of the proteins are crosslinked into the tertiarystructure.

“Insoluble proteins” refers to any protein which does not dissolve in aparticular aqueous environment such as in the cytoplasm of themicroorganisms producing the protein or the initial homogenizationmedium. Functionally defined these proteins are those proteins that canbe pelleted by standard ultracentrifugation procedures. The host cellsare lysed in a suspension medium capable of maintaining the desiredproteins insoluble from which they are separated from soluble materialsby centrifugation. The general methods of isolation of insolubleproteins are known in art. See for example U.S. Pat. No. 4,512,922.Examples of insoluble proteins formed in microorganisms include,insulin, chymosin, somatotropins, crystal protein of Bucillusthuringlensis, fibroblast interferon, viral proteins, and tissueplasminogen activator.

“Insoluble forms of somatotropin” refers to precipitated forms ofheterologous growth hormones being produced by a transformed organism.It is a type of insoluble protein.

“Selective solubilization” refers to a step in the extraction andpurification procedure in which the host debris is solubilized and thedesired insoluble protein remains insoluble.

“Somatotropin” refers to mammalian, fish and avian growth hormones.Somatotropins include analogs of these proteins wherein there issufficient amino acid sequence identity to permit biologic activity tobe maintained. Such analogs include recombinantly produced somatotropinshaving the same amino acid sequences as the heterogeneous species foundin purified preparations from pituitary glands and analogs artificiallycreated by modification of somatotropin encoding DNA such as describedin European patent application 103,395.

“Transformed microorganism” refers to prokaryote or eukaryote singlecelled organism containing or hosting a plasmid artificially insertedinto the cell using techniques well known in molecular genetics.

“Unbuffered alkaline solutions” refer to aqueous solutions having a pHabove 7.0 and containing nonbuffering salts such as potassium or sodiumhydroxide. Chelating agents such as ethylenediaminetetraacetic acid[EDTA] are optionally included. However, salts having significantbuffering capacity are not added to these solutions. These solutions areused to suspend cells prior to lysis and retrieval of desired insolubleprotein fractions.

DETAILED DESCRIPTION

The disclosed multistep procedure is optimally useful where the desiredproteins are insoluble. Although the process was developed to isolateand purify insoluble heterologous proteins, the kill step of the processhas general application to all insoluble proteins.

It is sometimes helpful to determine the proportion of desired proteinthat is in an insoluble form. In the case of heterologous proteinsproduced in transformed microorganisms strain variation may affect theproportion of insoluble to soluble protein. To determine the ratio ofsoluble to insoluble somatotropin as well as a comparison of yieldbetween strains, radioimmunoassay is best. Cells producing somatotropinsare lysed and labeled antibodies to the somatotropin which are added.The mixture is centrifuged and a comparison of the amount of label inthe supernatant versus the pellet gives the ratio insoluble to solublesomatotropin being produced by the organism. U.S. Pat. No. 4,512,922,columns 25-26.

The various steps for isolation of somatotropins from transformedmicroorganisms are an effective cell kill, cell disruption, selectivesolubilizing or removal of undesired cellular debris, solubilizing ofthe somatotropin followed by further purification by filtration ordialysis and combinations thereof. Each step can be achieved in anynumber of ways known in the art.

Use of cell kill means are optional prior to cell disruption and severalmeans are known in the art. Cell kill is useful from a safetyperspective. In this invention cell kill is accomplished with anon-polar organic solvent which will minimize protein precipitation. Thepreferred solvents are toluene or xylene. The amount of solvent isdependent upon the concentration and type of cell being killed. It isdetermined empirically using standard viability tests. Ordinarily alethal dose is defined as that concentration of solvent necessary toreduce the number of viable cells in a 10 ml sample to less than one.Viability is readily determined by culture techniques which varyaccording to the organism being exposed to the solvents. Thesetechniques are known to those of ordinary skill in the art. Generally,it is necessary to add amounts of solvent beyond the saturation point ofthe aqueous layer. Amounts in excess of the minimum lethal dose are notharmful to the process although containment of inflammable fumes must betaken into account to ensure adequate safety.

After killing the microorganisms, the cells are disrupted by any numberof known means, such as by sonication, pressure, or detergent. Thepreferred method-will not heat the solution and yield high levels ofcellular rupture. The preferred method is to use a homogenizer such as aManton-Gaulin to disrupt the cells. A second cycle of disruption is alsohelpful.

The disrupted cells are suspended in a buffer of low ionic concentrationunder alkaline conditions or in deionized water at a pH of 8-10.5. Thesolution is kept between 2°-15° C. and immediately treated withdetergents that selectively solubilized cell membranes and otherundesired cellular debris while not solubilizing the somatotropins.Effective detergents for rbSt include: Triton X-100 and deoxycholate,with Tergitol 15-5-7 being preferred. Sodium borate is the preferredbuffer.

The solubilized cell debris is mechanically separated from insolublesomatotropin by centrifugation sufficient to sediment the solids. Thesediments are then repeatedly suspended in the above buffer andrecentrifuged to eliminate the solubilized cellular debris.

The Insoluble somatotropins are next solubilized and oxidized undercontrolled conditions. The conditions are designed to minimizeundesirable polymerization which would reduce yields.

Using SDS or a detergent of formula I wherein n is 8 through 20inclusive; R₁ is methyl or ethyl; and R₂ is hydrogen, ethyl, methyl orn-propyl, solubilization and oxidation of somatotropins can be achievedat relatively high concentrations. The ratio of somatotropin todetergent is dependent on the detergent's acid moiety: the weaker theacid moiety of the detergent, the greater quantity of detergent requiredfor solubilization. For example, using sodium dodecyl sulfate which hasa relatively strong acid moiety, a ration of 1:1 is sufficient. However,using detergents of formula I, ratios of 2-5 are preferred. Use of abuffer to dilute the detergent is optional.

Once the somatotropin is solubilized, the oxidation is optionallypromoted by introducing air through the mixture. Air is introduced byeither agitation or passing air directly through the solution. The rateand method of introduction of air is dependent on the quantity of thepreparation. It is necessary to allow oxidation to go to completion toavoid downstream polymerization reagent during concentration. Oxidationcan be monitored by use of Ellman's reagent. Methods of Enzymology,25:457-464 (1972).

The preferred method for solubilizing rbSt is to add an aqueous solutionof N-Lauroyl methylglycine (Hamposyl R L-95, W. R. Grace, Lexington,Mass.) in a sodium borate buffer at 0.1-0.5 M and pH 8-10.5. The amountof Hamposyl needed to solubilize rbSt is approximately 2-10 g for eachgram of rbSt present with a 4:1 ratio being preferred. Less detergentreduces the solubility of rbSt and more detergent will increaseproduction costs. The solution is then agitated at 15-25° C. while airis passed through the mixture for at least 16 hours.

The detergent is then removed by anion exchange resin. The preferred ionexchange resin is Dowex 1×4 in the chloride form (Dow Chemical Co.). Theresin is removed by filtration and fresh resin added and removed untilthe mixture is essentially detergent free. Upon removal of detergent,the rbSt is split into two populations, monomers and large polymericbodies. The polymeric material forms a precipitate which is readilyremovable as described in the next step. The remaining somatotropin isin the monomeric and native form.

The detergent-free mixture is further purified by removal of remainingsolid contaminants including polymeric rbSt. Said contaminants areremoved by techniques well-known in the art, such as centrifugation ormicrofiltration. The preferred method is to use filter aids such, asCELATOM (Eagle-Picher, Cincinnati, Ohio) or SUPERCEL (The Johns-ManvilleCo., New York, N.Y.) which are added and removed by filtration. Thefiltrate may be passed through a microfilter having pore sizes of 0.45microns.

The filtrate is concentrated by ultrafiltration. The preferredfiltration membrane being 10,000 molecular weight cut-off polysulfonemembranes. The somatotropin which has a molecular weight of overapproximately 20,000 is retained and the retentate is dialyzed againstwater, pH 9-10 with base such as ammonium or sodium hydroxide. BecauserbSt is apt to form colloids at high concentrations which deposit on thefilter, it is helpful to pass alkaline water through the filteringdevice to avoid substantial losses at this point.

The somatotropin can be dialyzed and freeze dried at this point orfurther purified by passage through an ion exchange column, such as DEAEsepharose. The column is kept under alkaline conditions of 9.0-10.5.Fractions containing somatotropin are collected from the column.

The fractions are concentrated using ultrafiltration or other similarmethods and the concentrate filtered a final time through a microfilter.The polished permeate is then freeze dried.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. The following detailed example describes how toperform the processes of the invention and is to be construed as merelyillustrative, and not limitations of the preceding disclosure in any waywhatsoever. Those skilled in the art will promptly recognize appropriatevariations from the procedures as to reactants, conditions andtechniques.

EXAMPLES Example 1

Extraction of bSt from E. coli and Oxidation to the Native State.

Step 1. Killing E. coli with Toluene.

Transformed E. coli expressing bSt can be obtained from the AmericanType Culture Collection (ATCC No. 31826) or from other commonly knownsources. The cells are grown in a fermenter and harvested underconditions appropriate to turn on the particular promotor present, suchas described in European Patent Application 131 893 A1 for E. coli ATCC31826 or U.S. Pat. No. 4,511,503.

Ten kg of wet cells having a dry weight of 3.5 grams of cells andcontaining 50 mg rbSt/gm wet cells are placed into a container in whichthe oxygen is reduced to less than 5% through the introduction ofnitrogen while agitating the solution. This is a cell slurry of 200liters. To this slurry is added 6.7 liters of toluene and the slurry isagitated. for 1 hour at 15° C.

Step 2. Rupture of the Cells.

The dead cells are centrifuged with a Westfalia solids dischargingcentrifuge at 12,000×g at 4 lpm. The cell pellet is resuspended indeionized water to a total volume of 46 liters. Thirty-five grams ofEDTA is added and the pH adjusted to 9.0 with 1N NaOH. The cells areagitated until a uniform suspension is formed and cooled to 5° C. priorto homogenization in a Gaulin homogenizer. The Gaulin homogenizer isoperated at 9,000 psig. The ruptured cells are cooled to 5° C. as soonas possible and homogenized a second time at 10,000 psig. The product isthen cooled to 20° C. as soon as possible.

Step 3. Detergent and Buffer Washes.

To the suspension is added 2 liters of Tergitol 15-5-7. The solution isagitated for 30 minutes and centrifuged in the Westfalia solidsdischarging centrifuge (12000 g) at 2 lpm. The resulting pellet issuspended in 50 liters of deionized water and agitated for 15 minutesuntil uniformly suspended. The suspension is adjusted to a pH of 8-9with iN NaOH and recentrifuged in the Westfalia solids dischargingcentrifuge (12000 g) at 2 lpm.

Step 4. Solubilization of bSt and Oxidation.

The solid is immediately suspended in 50 liters of 100 mM sodium boratebuffer at pH 10.0 containing 1.5 kg of N-lauroyl methylglycine (HamposylL-95). The solution is vigorously agitated for 1 hr at 20° C. tocompletely solubilize the pellet and then gently agitated at 20° C.while adding air at 5 cubic feet per hr for at least 16 hours.

Step 5. Removal of Hamposyl with Dowex 1×4.

The pH of the Hamposyl extract is adjusted to 10.5 with 1N NaOH. Theextract is then fed at a rate of 400 ml/minute to a 75 liter column ofDowex 1×4 in chloride form. The column is washed with 45 liters of 50 mmsodium borate at pH 10.0 and the fractions containing rbSt arecollected.

Step 6. Polishing of the Hamposyl—Free Extract.

Three kg of standard Supercel are added to the above solution and thesolution is then agitated to obtain a uniform suspension. The mixture isthen filtered on a filter press. The resulting filter cake is washedwith 12 liters of 50 mM sodium borate at pH 10.0. The wash and thefiltrate are then combined.

Step 7. Ultrafiltration.

Using a 10,000 molecular weight cut off polysulfone membrane, thecombined filtrate and wash solutions from Step 6 are concentrated to 4liters. The concentrated solution is then diafiltered while maintaininga retentate volume constant using 40 liters of pyrogen-free water whichwas pH adjusted to 10.3 with 1N NaOH. The retentate is collected and theultrafilter washed with 6 liters of deionized water pH adjusted to 10.3.

Step 8. DEAE Sepharose Ion Exchange.

The rbSt concentrate is then fed through a column of DEAE sepharose incarbonate form. The sepharose is equilibrated with 5 mM sodium carbonateat pH 9.2. The concentrate is fed to the sepharose column at a rate of30 ml/minute and it is followed by 6 liters of 5 mM sodium carbonatebuffer at a pH of 9.2 at a rate of 300 ml/minute. The column is theneluted using 300 liters of 150 mM sodium carbonate buffer at pH 9.2,being fed to the column at a rate of 300 ml/minute. Fractions containingrbSt are saved.

Step 9. Ultrafiltration of the Eluate.

The fractions containing rbSt are adjusted to a pH of 10.0 using 1N NaOHand concentrated using a 10,000 molecular weight cut-off polysulphonemembrane to 4 liters. The 4 liters of solution are then diafilteredwhile maintaining a retentate volume constant using 40 liters ofpyrogen-free water that has been pH adjusted to 10.3 with NaOH. Theretentate is collected and the ultrafilter washed with 3 liters ofdeionized water pH adjusted to 10.3. The retentate and wash solutionsare then combined.

Step 10. Final Polish and Freeze Drying.

The retentate and wash solutions from Step 10 are concentrated to 400 mlusing a Pellicon R (Millipore Corp., Bedford, Mass.) filter containing 5square feet of a 0.45μ Durapore membrane. The concentrate is thendiafiltered while maintaining a constant retentate volume using 2.4liters of pyrogen-free water pH adjusted to 10.3 with NaOH. The permeateis then freeze dried.

1. A process for purifying an insoluble protein from a microorganismcomprising: 1) killing the microorganism by exposure to a lethal amountof an organic solvent consisting essentially of toluene; 2) lysing themicroorganism; and 3) recovering the insoluble protein.
 2. The processaccording to claim 1 wherein the insoluble protein is bovinesomatotropin or a bovine somatotropin analog.
 3. The process accordingto claim 2 wherein the microorganism is Escherichia coli.
 4. The processaccording to claim 1 wherein the microorganism is Escherichia coli.
 5. Aprocess for purifying an insoluble protein from a microorganismcomprising: 1) killing the microorganism by exposure to a lethal amountof an organic solvent consisting essentially of toluene and xylene; 2)lysing the microorganism; and 3) recovering the insoluble protein. 6.The process according to claim 5 wherein the insoluble protein is bovinesomatotropin or a bovine somatotropin analog.
 7. The process accordingto claim 6 wherein the microorganism is Escherichia coli.
 8. The processaccording to claim 5 wherein the microorganism is Escherichia coli.
 9. Aprocess for purifying an insoluble protein from a microorganismcomprising: 1) killing the microorganism by exposure to a lethal amountof xylene; 2) lysing the microorganism; and 3) recovering the insolubleprotein.
 10. The process according to claim 9 wherein the insolubleprotein is bovine somatotropin or a bovine somatotropin analog.
 11. Theprocess according to claim 10 wherein the microorganism is Escherichiacoli.
 12. The process according to claim 9 wherein the microorganism isEscherichia coli.